|[Previous] | [Next]|
Table of Contents
Why Do Genetics
More Basic Concepts
Mutagenesis in vitro
Effects of Mutations
Two Factor Crosses
Other Mapping Methods
Problem Set 1
Problem Set 2
VIII A. DEFINITIONS USED IN GENETIC MAPPING
Why do geneticists indulge in mapping? The answer to that question depends largely on the sorts of mapping that are employed because each level or type of mapping can answer certain questions. Because of this, we will treat the rationale behind mapping in two ways, one for gross Mapping and one for "fine structure" mapping. In either case, DNA is transferred into a recipient cell under conditions where there is a selection for the stable inheritance of the incoming DNA. Typically this involves a selection for recombination of the incoming DNA with a replicon in the recipient.
One performs gross mapping if one's intentions are to either place the marker of interest somewhere on a chromosomal map, or to find out any other relevant or irrelevant markers that happen to be genetically linked (see VIIID and E). This sort of mapping is often reported in the literature but, in general, it does not really tell you very much. Arguably, it just sets up the system for future strain constructions, allows preliminary genetic analysis of other mutations, helps in the construction of either R-primes or F-primes for complementation analysis, and allows some sort of comparison to genetically similar systems. For example, if you knew you had three loci (with a particular phenotype) and showed that they were each linked to a different selectable marker and unlinked to each other, then you have answered nearly all the interesting questions that can be addressed with this level of genetic analysis.
It is now becoming possible to do gross mapping physically. This has required the identification of restriction enzymes that cut very rarely (<20 times per genome) and the development of an electrophoresis system, orthogonal field electrophoresis, capable of resolving very large DNA fragments. The localization of a gene to a given fragment, using physical or genetic methods, provides gross, physical mapping information (section VIII E).
The goal of fine structure mapping is to order mutations, which are known to map in a given small region, into a one-dimensional array. This array actually says little about physical distance between the mutations, but a comparison of the order of mutations with the phenotypes that they cause allows strong statements to be made about the organization of the genetic system. Physical mapping can also order mutations and provide that ordering with actual physical distances; it will be considered in section VIII E. Properly, this array should be ordered with respect to other external markers. This ordering will allow you to make sense of your complementation data (you can then tell polarity from allelism); it allows the "clustering" of phenotypes that, in conjunction with complementation, helps define genes and gene functions; when performed in conjunction with "reversion analysis", it helps confirm that the mutation you are dealing with is a single and not a double mutation. Increasingly, the fine structure analysis of DNA is the only form of mapping of interest to molecular biologists, and deletion mapping is the best way to genetically perform such mapping. As sequencing methodology has become ever more rapid, it is becoming reasonable to map by sequencing, thus providing a physical reality to the mutation order. On the other hand, while mapping itself is becoming less relevant, the concept of linkage remains important and will be the focus of this section of the text.
It is important to understand the difference between mapping and complementation. Complementation is a test of function. It asks the question if two separate regions of DNA can supply all required functions for an apparently wild-type phenotype. Mapping is a test of sequence. It asks if, and with what frequency, two non-identical versions of the same genetic region are capable of recombining to generate a wild-type sequence. Complementation is therefore best analyzed in the absence of recombination while mapping typically demands recombinational events. Your mapping analyses have to be so devised that you can select for a phenotype that requires one or more recombinational events.
A term that is used with great frequency in discussions of mapping is linkage. Linkage is defined as the frequency with which two sites (a site can either be the site of a mutation or the site of the wild-type version of the mutation) on a piece of DNA are co-inherited using a particular gene transfer system. As such, it is a function of two variables: (1) The frequency with which the two sites are brought into the same cell by that particular gene transfer system (termed "end effects" in some of the following sections, in reference to the "ends" of the transferred DNA) and (2) the frequency with which they are both recombined into the chromosome. Another statement of the latter point is that, for linkage to be observed, the recombination events occur "outside" each site and not between them. Ignoring end effects, linkage is inversely proportional to the likelihood of a recombinational event occurring between two sites and (since recombination events are random and their likelihood increases with the increasing size of homologous regions available for recombination) therefore, to the distance between the sites:
The product strain (the genotypically altered recipient) of a recombinational event is often referred to as a recombinant.
Genetic mapping also makes the assumption that there is only one piece of DNA exchanged between the two organisms. Thus it is assumed that if two markers enter a recipient cell, they must be on the same piece of DNA and they therefore must be "linked" in that gene transfer system. If one utilizes a gene transfer system where more than one distinct piece of DNA can enter the same cell, one of the assumptions used in mapping is violated and problems in interpretation can occur since the apparent linkage would reflect the frequency of the two markers entering the same cell separately and not the genetic distance between them. This latter case can occur in either transformation or in generalized transduction with the highly efficient transducing phage P22HT, since these two systems are so efficient at moving DNA into a recipient that it is quite possible to get more than one piece of DNA into a given recipient. Such a phenomenon is known as congression.
The examples in the following sections ( section VIII B&C) arbitrarily concentrate on generalized transducing phage as the transfer system. Other transfer systems can be used and their use will be noted in section VIII F
|This page was last built with Frontier and Web Warrior on a Macintosh on Thu, Sep 21, 2000 at 1:01:27 PM.|